Interaction of G alpha(12) with G alpha(13) and G alpha(q) signaling pathways

Abstract

The G(12) subfamily of heterotrimeric G-proteins consists of two members, G(12) and G(13). Gene-targeting studies have revealed a role for G(13) in blood vessel development. Mice lacking the alpha subunit of G(13) die around embryonic day 10 as the result of an angiogenic defect. On the other hand, the physiological role of G(12) is still unclear. To address this issue, we generated G alpha(12)-deficient mice. In contrast to the G alpha(13)-deficient mice, G alpha(12)-deficient mice are viable, fertile, and do not show apparent abnormalities. However, G alpha(12) does not seem to be entirely redundant, because in the offspring generated from G alpha(12)+/- G alpha(13)+/- intercrosses, at least one intact G alpha(12) allele is required for the survival of animals with only one G alpha(13) allele. In addition, G alpha(12) and G alpha(13) showed a difference in mediating cell migratory response to lysophosphatidic acid in embryonic fibroblast cells. Furthermore, mice lacking both G alpha(12) and G alpha(q) die in utero at about embryonic day 13. These data indicate that the G alpha(12)-mediated signaling pathway functionally interacts not only with the G alpha(13)- but also with the G alpha(q/11)-mediated signaling systems.

Figures

Figure 1

Targeted inactivation of the murine Gα12 gene. (A) Genomic structure of murine Gα12 gene (WT), targeting construct (TC), and replaced mutant allele (Mut.), as described in Materials andMethods. (B) Western analysis of Gα12 mice. Cholate extracts from mouse brain were used for detection by Western analysis. An approximately 45-kDa band representing Gα12 was seen in wild-type tissue with antibodies against the N (N-20, Santa Cruz Biotechnology) and C termini (AS 233). Expression of Gα13 and Gαq was also examined in both wild-type and Gα12 mutant mice.

Figure 2

Embryos from offspring of Gα12±Gα13± intercrosses. (A) Embryos taken at E8.25. (B) Whole-mount staining of E9.5 embryos with antiplatelet–endothelial cell adhesion molecule-1 antibody (PharMingen).

Figure 3

LPA-induced cell migration. GFP, Gα12, and Gα13 expressing Gα13−/− and Gα12−/−Gα13−/− cell lines were established by using the retroviral expression system as described inMaterials andMethods. Migration experiments were performed in triplicate by using 24-well Transwell migration chambers, and migrated cells were quantitated in five random fields and expressed as a percentage of the control cells (−) that are always run at the same time. Two different G13−/− lines and two different G12−/−G13−/− lines were examined and showed similar results in at least two independent experiments.

Figure 4

Embryos from offspring of Gαq±Gα12± intercrosses. (A) Embryos taken at E12.5. (B) Embryos taken at E13.5.